PROJECT SUMMARY Malaria kills an estimated one million people a year, most of them children. While there is a vaccine nearly licensed, it only induces limited and short-lived protection. A major problem in the development of an effective vaccine is the poor understanding of the mechanisms that generate protective immunity. In malaria, CD4 T cell IFN-?+ responses, called T helper type 1 (Th1), are necessary to control parasite growth. On the other hand, IL- 21-producing T follicular helper (Tfh) cells are essential for generating high-affinity antibody responses for parasite clearance. Whereas the transcription factors Bcl6 and STAT3 regulate the development of Tfh cells, T-bet directs the differentiation and commitment of Th1 cells. Here, we will use the murine parasite Plasmodium chabaudi, which causes a form of chronic malaria that accurately mimics human malaria, to gain knowledge regarding the design of protective immune responses. Our previous data shows that there is a mixed Th1/Tfh response in P. chabaudi malaria infection and that T-bet and Bcl6 co-localize to the nucleus of Th1 cells in this chronic response. As Bcl6 is downstream of STAT3 and can inhibit the Th1 master regulator, T-bet, and induce Tfh characteristics, this implies that the balance of humoral and cellular responses are regulated by these molecules. Moreover, STAT3 is also downstream of pathology-regulating cytokines. Therefore, we hypothesize that immune control of malaria parasitemia and severity of disease outcome, are regulated by the balance of Th1 and Tfh characteristics at the peak of infection. Phenotypic and functional profiling of CD4 T cells show that mixed Th1/Tfh cells constitute the majority of the response in other chronic infections as well. In order to investigate the molecular determinants of this phenotype, we will use T cell-specific Bcl6- and STAT3-deficient mice to study the generation and flexibility of Th1 cells during the peak of infection. The first aim will determine the effect of deletion of these transcription factors in T cells on peak parasitemia and pathogenesis in vivo. We will also test the role of these factors in generation of parasite-specific antibodies and parasite clearance in the later stages. The second aim will address the molecular basis of flexibility in chronically-stimulated mixed phenotype Th1/Tfh cells. We will examine the phenotype of T cells in infected deficient animals to determine the role of each factor in the balance of humoral and cellular immunity. Then we will test the commitment of in vitro generated Bcl6- and STAT3-deficient Th1 cells on adoptive transfer into infected hosts. Polychromatic flow cytometry and Real Time PCR will be used to measure the expression of Th1 and Tfh markers in CD4 T cells at the peak of infection. The objective of this project is to elucidate the lineage, maintenance, and in vivo function of this chronically-stimulated Th1/Tfh population with potential implications for vaccine strategies. The execution of this project with the assistance of my sponsor and co-sponsors will contribute to my training as an independent scientist and prepare me to add to the study of infectious disease immunology as an academic scientist.